Selective ablation of thymic and peripheral Foxp3+ regulatory T cell development

Foxp3+ regulatory T (Treg) cells of thymic (tTreg) and peripheral (pTreg) developmental origin are thought to synergistically act to ensure immune homeostasis, with self-reactive tTreg cells primarily constraining autoimmune responses. Here we exploited a Foxp3-dependent reporter with thymus-specific GFP/Cre activity to selectively ablate either tTreg (ΔtTreg) or pTreg (ΔpTreg) cell development, while sparing the respective sister populations. We found that, in contrast to the tTreg cell behavior in ΔpTreg mice, pTreg cells acquired a highly activated suppressor phenotype and replenished the Treg cell pool of ΔtTreg mice on a non-autoimmune C57BL/6 background. Despite the absence of tTreg cells, pTreg cells prevented early mortality and fatal autoimmunity commonly observed in Foxp3-deficient models of complete Treg cell deficiency, and largely maintained immune tolerance even as the ΔtTreg mice aged. However, only two generations of backcrossing to the autoimmune-prone non-obese diabetic (NOD) background were sufficient to cause severe disease lethality associated with different, partially overlapping patterns of organ-specific autoimmunity. This included a particularly severe form of autoimmune diabetes characterized by an early onset and abrogation of the sex bias usually observed in the NOD mouse model of human type 1 diabetes. Genetic association studies further allowed us to define a small set of autoimmune risk loci sufficient to promote β cell autoimmunity, including genes known to impinge on Treg cell biology. Overall, these studies show an unexpectedly high functional adaptability of pTreg cells, emphasizing their important role as mediators of bystander effects to ensure self-tolerance.


Introduction
The discovery of genetic Foxp3 gene mutations as the culprit of the fatal autoimmune syndrome in the spontaneous scurfy mouse mutant (1,2) and human IPEX patients (3,4) provided the basis for unraveling the key role of Foxp3 + regulatory T (Treg) cells in dominant immunological tolerance.Observations in Foxp3 genetargeted mice further corroborated Treg cell paucity as the primary cause of early death and multi-organ autoimmunity in Foxp3deficient mice (5) but also revealed the peripheral accumulation of Treg cell-like 'wanna-be' CD4 + T cells with self-reactive specificities (6)(7)(8) that contribute to the disease pathology (9,10).Acute Foxp3 + Treg cell ablation recapitulated some, but not all aspects of the scurfy syndrome in non-autoimmune-prone mice (11,12) and highlighted the continuous requirement of Treg cells to constrain organ-specific autoimmune responses in the spontaneous non-obese diabetic (NOD) mouse model of human type 1 diabetes (T1D) (13).
Since then, it has become clear that the physiologic Treg cell pool is developmentally heterogeneous (14)(15)(16), consisting of intrathymically (tTreg) and peripherally (pTreg) induced Treg cells that originate from distinct CD4 + CD25 high Foxp3 -precursor cells residing in thymus (17) and peripheral lymphoid tissues (18), respectively.In the thymus, distinct CD4 + CD8 -single-positive (CD4SP) precursor cells, which exhibit low levels of Foxp3 protein preceding the up-regulation of CD25 expression, further expand the mature tTreg cell repertoire (19).In early studies examining the functional specialization of Treg cell developmental subsets by adoptive transfer immunotherapy of newborn scurfy mice (20), total Foxp3 + Treg cells prevented disease lethality, but did not suppress chronic inflammation and autoimmunity, which required the provision of Foxp3-sufficient CD4 + T cells to facilitate the extrathymic conversion of initially Foxp3 -T cells into functional Foxp3 + Treg cells (20).According to the prevailing view, tTreg cells are primarily positively selected by self-antigens during intrathymic development and are functionally specialized to control immune homeostasis and autoimmune responses (14,21).The tTreg cell compartment in the spleen (SPL) and lymph nodes (LNs) has also been proposed to harbor Foxp3 + ST2 + common precursors for tissuetype Treg cells (22) that accumulate and perform homeostatic and regenerative functions in nonlymphoid tissues (23), such as the visceral adipose tissue (24,25).Consistent with tTreg cells as primary regulators of autoimmune responses, studies in mice with Foxp3 gene-targeted deletion of conserved non-coding region 1 (CNS1) (Foxp3.CNS1 -/-), which exhibit a significant, albeit incomplete block of pTreg cell development (26), failed to reveal severe autoimmune symptoms and have implicated pTreg cells in the control of immune responses at mucosal surfaces (27) and maternalfetal tolerance (28).More recently, pTreg cells dependent on the gut microbiota have been shown to mediate functions beyond dominant suppression by facilitating muscle regeneration (29).With regard to a putative role of pTreg cells in the control of autoimmune responses, previous studies in the NOD model showed that dendritic cell (DC)targeted self-antigen can encourage highly diabetogenic CD4 + Foxp3 - T cells to acquire a Foxp3 + pTreg cell phenotype (30, 31), and that naturally induced, b cell-reactive pTreg cells are superior to tTreg cells with the same T cell receptor (TCR) specificity in constraining the manifestation of overt diabetes in a NOD.Rag1 -/-adoptive transfer model (32).While Foxp3-deficient NOD mice failed to develop insulitis and overt diabetes (33), studies in Foxp3.CNS1 -/- NOD mice have provided ambiguous results, providing evidence for either a dispensable (34) or nonredundant function (35) of Foxp3.CNS1-dependent pTreg cells in the control of destructive b cell autoimmunity.In these studies, the relative contribution of tTreg cells to autoimmune b cell protection has not been directly addressed, owing to the lack of mouse models with selective tTreg cell paucity.
Here, we have exploited tTreg cell lineage-specific GFP/Cre recombinase activity in dual Foxp3 RFP/GFP reporter mice (32,36) to generate complementary mouse lines that are deficient in either the tTreg (37) or pTreg cell lineage, while sparing the respective sister population.The results of subsequent loss-of-function studies revealed an unexpectedly high functional adaptability of naturally occurring pTreg cells in mice with selective tTreg cell paucity, thereby preventing the manifestation of severe scurfy-like symptoms commonly observed in mice with complete Treg cell deficiency.However, the acquisition of an increased genetic autoimmune risk associated with compromised Treg cell activity unleashed high mortality and a distinct pattern of autoimmune diseases, including severe b cell autoimmunity and overt diabetes.
Mice were considered diabetic at blood glucose levels above 200 mg/ dl on at least two consecutive measurements or with blood glucose levels once above 400 mg/dl.All mice were housed and bred at the Animal Facility of the CRTD under specific pathogen-free conditions.Animal experiments were performed as approved by the Landesdirektion Dresden (25-5131/502/5, TVA 5/2020; 25-5131/522/43, TVV41/2021).

Histopathology
After euthanizing the mice using CO 2 inhalation, organs were collected and briefly washed in PBS.Subsequently, the tissues were fixed in a 4% paraformaldehyde solution (Sigma-Aldrich), paraffinembedded, and 5 µm sections were cut.These sections were then stained with hematoxylin and eosin to assess histopathological changes.A total of 13 organs were examined (lung, heart, thymus, thyroid gland, stomach, liver, intestine, kidney, pancreas, urinary bladder, mesenteric adipose tissue, reproductive tract, brain) in a blinded manner to evaluate the presence and extent of inflammation and necrosis (none: 0; mild: +; moderate: ++; severe: +++) as described elsewhere (40).

Genomic PCR-based Idd gene analysis
Genomic DNA was isolated from tail biopsies using the NucleoSpin DNA RapidLyse kit (Macherey-Nagel) according to the manufacturer's protocol.Genomic PCR was performed using DreamTag green DNA polymerase and buffer, dNTPs (all Thermo Fisher, Life Technologies), a set of 36 primer pairs (Eurofins Genomics) that cover most of the known Idd loci (see Supplementary Materials and Methods for a complete list) (41), and a Biometra Trio Thermocycler (Analytik Jena).
In Foxp3 RFP/GFP x R26 DTA mice (hereafter referred to as DtTreg mice) (37), GFP/Cre recombinase activity induces DTA expression selectively in the tTreg cell lineage by excision of an upstream loxPflanked STOP cassette (38) (Figure 1B).DTA-mediated ablation (≥ 99.8%) resulted in the absence of RFP + GFP + tTreg cells in scLN (Figure 1D) and at other peripheral sites, such as mesenteric LN and SPL (Figure 1E).A similarly high ablation efficiency was observed in peripheral lymphoid tissues of DtTreg mice with heterozygous (B6.R26 DTA ) and homozygous (B6.R26 DTA/DTA ) expression of the R26.STOP-DTA transgene, and over a wide age range (3-80 weeks; data not shown).As compared to Foxp3 RFP/GFP mice, selective tTreg cell paucity had no appreciable impact on the proportional distribution of CD4 + and CD8 + T cells in scLNs (Figure 1D, left), but was accompanied by a > 6-fold increase in the percentage of RFP + GFP -pTreg cells among CD4-gated T cells (Foxp3 RFP/GFP mice: 1.6 ± 0.5%; DtTreg mice: 10.5 ± 1.0%) (Figures 1D, E; see Supplementary Figure S1 for CD4 + T cell and Treg cell numbers).This marked increase in the pTreg cell population size of DtTreg mice largely compensated for the numerical impairment of the overall Treg cell pool in peripheral lymphoid tissues (Figure 1E; Supplementary Figure S2A), but to a significantly lesser extent in peripheral blood (Supplementary Figure S2B).In aged, > 1-year-old DtTreg mice, pTreg cells even exceeded total Treg cell frequencies of age-matched Foxp3 RFP/GFP mice (Supplementary Figure S2C).
To obtain mice with selective pTreg cell paucity (hereafter referred to as DpTreg mice), the Foxp3 IRES-RFP reporter of Foxp3 RFP/GFP mice was replaced by breeding with a Creactivatable Foxp3.STOP cassette (Figure 1C).Hemizygous and homozygous Foxp3.STOP mice succumb to severe scurfy disease due to the absence of functional Foxp3 protein and Treg cells (43).In DpTreg mice, BAC.Foxp3 GFP/Cre -mediated activation of Foxp3 expression selectively in tTreg cell lineage-committed thymocytes allowed for the formation of a robust peripheral GFP + tTreg cell compartment (Figures 1D, E; Supplementary Figure S1B), while the extrathymic generation of pTreg cells remained precluded by the Foxp3.STOP cassette.Consistently, in peripheral lymphoid tissues of DpTreg mice, the expression of Foxp3, Helios, and Nrp1 was absent in FACS-purified CD4 + GFP -cells, but readily detectable in CD4 + GFP + tTreg cells (Figure 1F).Interestingly, selective pTreg cell deficiency resulted in a sustained reduction of the peripheral tTreg cell pool in adult (Figure 1E; Supplementary Figure S2A) and aged (Supplementary Figure S2C) DpTreg mice, consistent with previous observations in Foxp3.CNS1 −/− mice with impaired pTreg cell development (26,27).These data, in conjunction with the ability of pTreg cells to compensate for the tTreg cell loss in DtTreg mice, suggest that pTreg cells are subject to less stringent constraints of the T cell receptor (TCR)-dependent clonal niche in peripheral lymphoid tissues, as compared to tTreg cells (44)(45)(46).
Next, we extended our observation of normal T lymphopoiesis in the majority of DtTreg mice to the analysis of B lymphopoiesis in the adult bone marrow (BM).In BM of scurfy mice, T cell-mediated autoimmune responses and systemically elevated levels of inflammatory cytokines have been shown to cause a complete block of B cell development, which is reflected by the absence of early Pro/Pre-B-I cell precursors and newly formed IgM + B cells (47)(48)(49)(50).However, our comparative flow cytometric analyses failed to reveal evidence for dysregulated B lymphopoiesis in DtTreg and DpTreg mice, as compared to Treg cell-proficient Foxp3 RFP/GFP mice (Supplementary Figure S3).This included comparable BM and SPL cellularity (Supplementary Figure S3A), as well as proportions and numbers of early B220 + c-kit + Pro/Pre-B-I precursor cells and immature B220 low IgM + B cells in BM (Supplementary Figures S3B,  C), and of newly formed IgD low IgM high B cells in the SPL (Supplementary Figures S3D, E).

DTA-mediated tTreg cell ablation occurs prior to thymic exit
In the thymus of Foxp3 RFP/GFP reporter mice, tTreg cell lineage commitment induces the sequential expression of RFP and GFP in initially Foxp3 − CD25 + CD4SP cells (32,37).Specifically, the developmental progression of Foxp3 − CD25 + CD4SP cells first initiates the simultaneous up-regulation of Foxp3 and RFP protein (giving rise to CD25 + RFP + GFP − cells) (Figure 2D; left panel), which is then followed by the timely delayed up-regulation of GFP/Cre expression, giving rise to newly formed Foxp3 + CD25 + tTreg cells that are RFP + GFP + (Figure 2E; left panel).In DpTreg mice, flow cytometry revealed no adverse effects of selective pTreg cell paucity on tTreg cell development (Figures 2D, E; right panels) and numbers of newly formed Foxp3 + tTreg cells (Figure 2F).In the thymus of DtTreg mice, tTreg cell development proceeded to the CD25 + RFP + GFP − CD4SP stage (Figure 2D; middle panel), but subsequent upregulation of BAC.Foxp3 GFP/Cre reporter expression promoted DTAmediated induction of apoptosis prior to thymic exit of newly formed CD25 + RFP + GFP + tTreg cells (Figure 2E; middle panel; Figure 2F).Thus, the observed deficiency in RFP + GFP + tTreg cells at peripheral sites (Figures 1D, E) was already established within the thymus.Whereas, the proportional increase of CD4-gated RFP + GFP − cells in DtTreg mice (Figures 2D, E) could be attributed to the intrathymic accumulation of mature pTreg cells (Supplementary Figure S4A) that originated from peripheral lymphoid tissues recirculating to the thymus, as indicated by heterogeneous CD25 expression levels and a 'recirculating' CD62L − CD69 + CD44 high phenotype (Supplementary Figure S4B) (51, 52).

pTreg cells prevent the early manifestation of severe autoimmunity in DtTreg mice
In our B6 scurfy colony maintained under specific-pathogen-free (SPF) conditions, approximately 50% of mice succumb to premature death by 35 days of age due to fatal autoimmunity associated with Foxp3 deficiency, and no mice live beyond 50 days (50).Within 35 days after birth, DtTreg (Figure 3A) and DpTreg (Figure 3B) mice appeared overall healthy, showing no appreciable spontaneous mortality (Figures 3A, B) or other scurfy-like symptoms (scaliness and crusting of eyelids/ears/tail, hepatomegaly, splenomegaly, lymphadenopathy, etc.) (data not shown).Consistently, our analysis of genotype distribution among 5-week-old mice revealed an unbiased heredity of the DtTreg (Figure 3C) and DpTreg (Figure 3D) phenotype.
The impaired generation of pTreg cells in Foxp3.CNS1 −/− mice has been reported to impinge on maternal-fetal tolerance by increasing the resorption of semiallogeneic fetuses (28).Our data show that the number of viable offspring produced by syngeneic DtTreg pair mating did not significantly differ from that of Foxp3 RFP/GFP mice (Figure 3E).In contrast, interstrain breeding of DpTreg mice gave rise to significantly reduced numbers of offspring, correlating with the DpTreg phenotype of the breeding female (Figure 3F).These findings in DtTreg and DpTreg mice, in conjunction with impaired implantation of syngeneic embryos after maternal Foxp3 + Treg cell depletion (53,54), imply that pTreg cells may contribute to maternalfetal tolerance even in syngeneic pregnancy.
The unexpected absence of severe scurfy-like symptoms in young B6.DtTreg mice suggested that selective tTreg cell paucity can be largely compensated by increased pTreg cell numbers (Figures 1D, E).We next asked whether the observed pTreg cell behavior in the DtTreg model can be recapitulated in scurfy mice neonatally injected with CD4 + GFP -T cell populations (including RFP + GFP -pTreg cells) that had been FACS-isolated from peripheral lymphoid tissues of Foxp3 RFP/GFP mice for RFP + GFP + tTreg cell depletion (Figure 3G, bottom left).In these experiments, total CD4 + T cells (including RFP + GFP -pTreg and RFP + GFP + tTreg cells) were included for comparison (Figure 3G, top left).Both cohorts of scurfy recipients were viable (Figure 3H) and appeared phenotypically healthy until the end of the observation period, apart from mild symptoms of delayed growth and exfoliative dermatitis in individual mice that received tTreg cell-depleted CD4 + GFP -T cells (data not shown).In the absence of tTreg cells, the adoptive CD4 + GFP -T cell transfer resulted in a marked accumulation of RFP + GFP -pTreg cells among CD4 + T cells (day 0: 4.1%; day 25: 78.4 ± 2.6%) in scurfy recipients (Figure 3G, bottom panels), most likely due to both the proliferative expansion of preformed pTreg cells and the conversion of initially CD4 + Foxp3 -T cells (20).In scurfy recipients of total CD4 + T cells, RFP + GFP -pTreg cell frequencies among CD4-gated cells also increased (day 0: 3.5%; day 25: 20.7 ± 1.1%), but the initial pTreg:tTreg cell ratio of 1:3 was preserved (Figure 3G, top panels).Overall, these data indicate that pTreg cells can fill up the tTreg cell niche in both DtTreg (Figures 1D, E) and scurfy mice (Figures 3G, H) while maintaining a stable RFP + GFP -phenotype.

Maintenance of T cell homeostasis in adult DtTreg mice
Our initial characterization of young DtTreg mice failed to reveal evidence for disease symptoms associated with selective tTreg cell paucity.Consistently, mild immune infiltrations were limited to the salivary gland (mouse #1), and the lung (mouse #1) of individual DtTreg mice, but could not be observed in other organs, such as the liver or thyroid gland (Figure 4A).Interestingly, although B6.DtTreg mice maintained normoglycemia, histological analyses consistently revealed pronounced immune infiltrates in the pancreas (Figure 4A, bottom panels), which is in contrast to previous studies in other settings of tTreg cell deficiency, including Foxp3-deficient mice (33) and acute Treg cell ablation in the 'Depletion of Regulatory T cells' (DEREG) mouse model on the B6 and NOD genetic background (13).
With the advancing age of DtTreg mice, we noticed rare cases of spontaneous deaths, which became first apparent at the age of 7 weeks (R26 DTA : 18.7%, R26 DTA/DTA : 19.0%; Figure 4B).The mortality of DtTreg mice further increased thereafter, reaching a plateau by 10 weeks that was maintained until the end of the 20week observation period (R26 DTA : 25.5%, R26 DTA/DTA : 30.7%), remaining well below the high mortality of scurfy mice (Figure 4B).We rarely observed cases of spontaneous mortality among immunodeficient Rag2 -/-mice, or cohorts of tTreg cellproficient, BAC.Foxp3 GFP/Cre-DtTreg littermates, DpTreg mice, and Foxp3 RFP/GFP mice (Figure 4B).We further noticed that mortality appeared to be associated in part with a reduced body weight and thymic atrophy of the affected DtTreg mice (Figure 2B).Our subsequent analyses showed that the majority of DtTreg mice had a body weight corresponding to their age (85.3%), but also confirmed that individual mice failed to keep up with physiological body weight gain (Figure 4C), while the small and large intestine yielded an unsuspicious histopathological result (data not shown).
With regard to peripheral T cell homeostasis in adult DtTreg mice, total cellularity of scLNs (but not of mLNs and SPL; see also Supplementary Figure S3A) was moderately, although significantly increased, as compared to Foxp3 RFP/GFP and DpTreg mice (Figure 5A).We only occasionally observed adult DtTreg mice with pronounced lymphadenopathy and splenomegaly (< 10%; Figure 5A).The analysis of inflammatory cytokine production indicated moderately increased proportions of IFN-g-and IL-4producing CD4 + T cells (Figure 5B, top), and of IFN-g-producing CD8 + T cells (Figure 5B, bottom) in scLNs (but not in mLN or SPL; data not shown) of DtTreg mice.Increased expression of other cytokines (e.g., IL-2, IL-10, IL-17) could also not be observed (data not shown).Consistent with largely normal frequencies of CD4 + and CD8 + T cells in the majority of DtTreg mice (Figure 1D), our flow cytometric analysis of CD62L and CD44 expression revealed no evidence for systemically uncontrolled activation of CD4 + and CD8 + T effector cells, neither in scLNs (Figures 5C-E) or other peripheral lymphoid tissues, such as mLN or SPL (Figures 5D, E).

Compensatory adaptation of pTreg cell activity in the absence of tTreg cells
Despite its already high percentage share in steady-state Foxp3 RFP/GFP mice (32), flow cytometric immunophenotyping (Figure 6A) indicated that the percentage of pTreg cells with a CD62L low CD44 high effector/memory-like phenotype further increased in LNs of adult DtTreg mice (Figure 6B), which was in contrast to tTreg cells in DpTreg (Figure 6C) mice.Consistent with an overall activated phenotype, pTreg cells in peripheral lymphoid tissues of DtTreg mice exhibited significantly upregulated expression of CD25 and several other 'Treg cell signature' proteins with functional relevance (including CD103, ICOS, ST2, and KLRG1), as compared to pTreg cells from Foxp3 RFP/GFP mice (Figure 6D; Supplementary Figure S5, top panels).In contrast, the reduced accumulation of pTreg cells in peripheral blood of DtTreg mice (Supplementary Figure S2B) was accompanied by low levels of CD25 expression (Figure 6D; left panel).Additionally, tTreg cells of DpTreg mice exhibited neither an increased effector/memory-like compartment (Figure 6C) nor up-regulated 'Treg cell signature' protein expression (Figure 6D; Supplementary Figure S5, bottom panels), as compared to their tTreg cell counterparts in Foxp3 RFP/ GFP mice.Consistently, pTreg cells isolated from DtTreg mice suppressed the activity of Tresp cells more efficiently in standard cocultures, as judged by the inhibition of Tresp cell proliferation and CD25 expression, and as compared with tTreg cells from DpTreg mice or total Treg cells from Foxp3 RFP/GFP mice (Figures 6E, F).In summary, in contrast to tTreg cells of DpTreg mice, pTreg cells in peripheral lymphoid tissues (but not blood) of DtTreg mice acquire a highly activated state and increased suppressor function, which is indicative for their active involvement in constraining chronic immune dysregulation in the absence of tTreg cells (Figure 4).

Fatal autoimmune pathology in DtTreg mice on a mixed (B6>NOD) background
Our characterization of DtTreg mice on the B6 background revealed neither early-or late-onset of severe morbidity nor other signs of fatal autoimmunity (Figures 3A, 4A, B) consistently observed in mice with complete Foxp3 + Treg cell deficiency (2,5).However, the severity of autoimmune pathology associated with Treg cell deficiency can be markedly shaped by genetic factors: B6 scurfy mice can survive for up to 9 weeks after birth (Figure 4B), which is significantly longer than scurfy mice on the BALB/c background, all of which succumb to death within < 5 weeks of birth (20).Here, we aimed to explore how increased genetic susceptibility impinges on the survival and immune homeostasis of DtTreg mice by backcrossing the B6.DtTreg mouse line to autoimmune-prone NOD mice carrying the dual Foxp3 RFP/GFP reporter.On a pure NOD background, spontaneous T1D manifestation is under polygenic control of more than 20 insulindependent diabetes (Idd) gene loci (55), which additionally confer a broad susceptibility to multiple other autoimmune syndromes (peripheral neuropathy, autoimmune thyroiditis, etc.), albeit often with a low incidence (56).The analysis of (B6>NOD) hybrid mice obtained by two consecutive backcrosses (F2) revealed that selective tTreg cell paucity drastically decreased the survival of F2 DtTreg mice (both I-Ag7 +/-and I-Ag7 +/+ ; see below) to ≤ 20% within 20 weeks after birth (Figure 7A), as compared to F1 DtTreg mice (84.2%; Figure 7A) and DtTreg mice on a pure B6 background (R26 DTA : 74.5%, R26 DTA/DTA : 69.3%; Figure 4B).Male and female F2 DtTreg mice showed no significant differences in mortality (data not shown).Continued backcrossing further exacerbated morbidity, such that none of the F3 DtTreg mice lived beyond 14 weeks (Figure 7A).
In order to account for possible variability of disease pathology on a mixed (B6>NOD) background, we produced independent cohorts of F2 and F3 DtTreg mice originating from unrelated (B6>NOD) backcross breedings and parental (B6>NOD)F1 mice (Figures 7B-D).When we monitored the DtTreg offspring for scurfy-like symptoms, we found that the high incidence of spontaneous mortality depicted in Figure 7A was consistently accompanied by signs of distinct, partially overlapping autoimmune diseases in independent cohorts of F2 DtTreg mice (Figures 7C, D).Most prominently, we observed signs of wasting disease (WD; reduced body weight and size, failure to thrive), autoimmune diabetes (hyperglycemia), and peripheral neuropathy (PN; hindlimb paralysis).In the (B6>NOD)F3 generation, ≥ 90% of DtTreg mice suffered from either WD (62.2%),T1D (32.4%), and/or PN (10.8%) (Figure 7B).Histopathological analyses indicated massive immune infiltrates of the salivary glands, the lung, the stomach, the thyroid glands, and the pancreas associated with severe tissue damage predominantly affecting thyroid glands and pancreatic islets of NOD.DtTreg F3 mice, which correlated with their hyperglycemic state (Figure 7E; top panel).A more detailed analysis of pancreatic islets (10 -20 islets/mouse) revealed hardly any signs of immune infiltrates (mouse #1: 0%; mouse #2: < 10%; both non-diabetic) into the islets of tTreg cell-proficient (B6>NOD) F3 mice, whereas the majority of islets of DtTreg (B6>NOD)F3 mice (#1: > 90%, diabetic; #2: 70 -80%; nondiabetic; #3: > 90%, diabetic) showed marked immune infiltrates (Figure 7E; data not shown).In these experiments, other organs commonly targeted by severe autoimmune responses in Foxp3-deficient mice showed no or only minimal immune infiltrates in NOD.DtTreg F3 mice, such as the small intestine or the liver (data not shown).Notably, severe salivary gland autoimmunity could be observed in both DtTreg and Foxp3 RFP/GFP mice (Figure 7E; left panels), and thus driven by the increased genetic autoimmune risk of the (B6>NOD)F3 background, rather than selective tTreg cell paucity.Other hallmarks of the fatal autoimmune syndrome affecting Foxp3deficient mice could not be observed, such as skin lesions or scaliness and crusting of the eyelids, ears, and tail (data not shown).Throughout the present study, the manifestation of WD, T1D, and/or PN in tTreg cell-proficient littermates of (B6>NOD) hybrid DtTreg mice has also not been observed (Figures 7B-E, 8A; and data not shown).

Pancreatic b cell autoimmunity in (B6>NOD) hybrid DtTreg mice
While Foxp3 + Treg cell-deficient NOD mice fail to develop insulitis and overt diabetes (33), the data depicted in Figure 7 provided the first indications that selective tTreg cell paucity can promote severe insulitis and overt diabetes (32.4%;Figures 7B-E) in both males and females, despite incomplete backcrossing onto the NOD background.However, more definite conclusions on the role of tTreg cells in controlling pancreatic b cell autoimmunity were hampered by the overall early onset of high morbidity and mortality (Figure 7A).In fact, three diabetic F3 NOD.DtTreg mice additionally exhibited signs of WD and PN (Figure 7B), suggesting that some (B6>NOD) hybrid DtTreg mice may succumb to death before the diabetes diagnosis.We therefore tracked blood glucose levels in cohorts of 3-week-old, initially normoglycemic F2 NOD.DtTreg mice that showed no signs of WD, PN, or other scurfy-like symptoms (Figure 8A).In our colony of conventional NOD mice, the first diabetes cases become apparent at approximately 12 weeks of age and continuously increase to an incidence of 70-90% in females and 0-20% in males within 30 weeks of age (13,57).Whereas in (B6>NOD)F2 DtTreg mice, selective tTreg cell paucity unleashed a particularly severe form of T1D: > 50% of males rapidly progressed to overt diabetes within < 8 weeks after birth (Figure 8A), despite the usually observed female sex bias and kinetics difference in the NOD model (58).Flow cytometry-based MHC class II haplotyping indicated that diabetes manifestation in F2 DtTreg mice correlated with homozygous expression of the diabetogenic MHC class II molecule I-Ag7 of the NOD genetic background (Idd1), whereas mice co-expressing I-Ab of B6 origin remained normoglycemic during the observation period (Figure 8A).In contrast, high mortality (Figure 7A) and the manifestation of WD (Figure 7D) was independent of homozygous I-Ag7 expression.
In line with the absence of severe systemic autoimmune responses, pLNs of F2 DtTreg mice showed clear signs of lymphadenopathy, whereas the size of non-draining LNs (scLNs, mLNs) and SPL did not significantly differ between tTreg cell-deficient and -proficient (B6>NOD)F2 mice (Figure 8B).Consistently, numbers of CD8 + and CD4 + T cells (Supplementary Figure S6A, top panels) were selectively increased in pLNs of F2 DtTreg mice.
Consistent with our data in B6.DtTreg mice (Figures 1D, E), efficient intrathymic tTreg cell ablation in (B6>NOD)F2 DtTreg mice (Supplementary Figure S6B) was accompanied by a significant, up to 5.5-fold increase in the percentage of RFP + GFP - pTreg cells among CD4-gated T cells in peripheral lymphoid tissues (Figures 8C, D; see Supplementary Figure S6A for Treg cell numbers).Howeve r, in c ont ras t t o B6.D t Tr eg mic e (Supplementary Figure S2A), the population size of pTreg cells in (B6>NOD)F2 DtTreg mice only partially compensated for the numerical impairment of the overall Treg cell pool in the absence of tTreg cells (Figure 8D; Supplementary Figure S6A, bottom).Additionally, thymic cellularity (Supplementary Figure S6C) and numbers of T cell developmental stages (Supplementary Figure S6D) were consistently reduced in (B6>NOD)F2 DtTreg mice, as compared to in (B6>NOD)F2 Foxp3 RFP/GFP mice, probably due to increased hyperglycemia-induced stress and/or systemically elevated inflammatory cytokine levels.Contribution of NOD Idd loci to diabetes in (B6>NOD) hybrid DtTreg mice We found that the manifestation of overt diabetes was restricted to I-Ag7 +/+ (B6>NOD) hybrid DtTreg mice (Figures 7B-E, 8A), consistent with the requirement of Idd1 homozygosity for high penetrance of diabetes susceptibility in the NOD model (59).In fact, Idd1 was shown to confer most of the diabetes risk (60), but not to be sufficient to precipitate diabetes in Foxp3 + Treg cell-proficient NOD mice (60).We hypothesized that the early manifestation of diabetes in I-Ag7 +/+ F2 DtTreg males with high penetrance (Figure 8A) was driven by the acquisition of one or more additional, non-MHC-linked Idd loci.As expected, after only two backcross generations, PCR-based genomic Idd gene analysis (Supplementary Figure S7) indicated that the majority of Idd genes included in our survey was dispersible for diabetes development in I-Ag7 +/+ F2 DtTreg mice (Idd2, Idd3/Idd10/Idd17/ Idd18, Idd4, Idd14, Idd15) (Supplementary Figure S7A).This included Il2 gene polymorphisms (encoded by Idd3), which play an important role in the reduced IL-2 receptor signaling strength received by Treg cells in conventional, tTreg cell-proficient NOD mice, resulting in their functional deficiency (61, 62).
In addition to Idd1, a set of 5 Idd gene loci (Idd5.1,Idd7, Idd8/ Idd12, Idd9.1/.2, Idd13.3) was detectable in ≥ 80% of diabetic F2 DtTreg mice (Figure 8E), but was not sufficient to promote diabetes in tTreg cell-proficient I-Ag7 +/+ (B6>NOD)F2 littermates (Figure 8A; and data not shown).Interestingly, this rather small set of Idd loci was primarily characterized by harboring genes with well-known functions in the development, survival/maintenance, function of pancreatic b cells [Idd7, Idd8, Idd12 (63-67)] and Overall, these findings in DtTreg mice are consistent with a scenario, in which pTreg cell-mediated maintenance of immunological tolerance to pancreatic b cells can be abrogated by the acquisition of a limited set of Idd loci, some of which unfold their diabetogenic activity directly in pTreg cells.In support of this interpretation, comparative flow cytometry-based immunophenotyping revealed a correlation of some detected Idd loci and differential protein expression in pTreg cells of F2 DtTreg mice, as compared to pTreg cells in tTreg cell-proficient Foxp3 RFP/ GFP littermates (Figure 9).This included the absence of Idd3 (including Il2) and markedly increased expression levels of CD25 on pTreg cells from pLNs of diabetic F2 DtTreg mice, as compared to F2 Foxp3 RFP/GFP (Figures 9A, C; left panels).Relevant to their high diabetes susceptibility, the acquisition of Idd5.1 (68) by F2 DtTreg mice correlated with increased expression levels of ICOS (Icos) and PD-1 (Pdcd1) on pTreg cells (Figures 9A, C), and the accumulation of an unusual ICOS + PD-1 high pTreg cell subset in F2 DtTreg mice, but not in Foxp3 RFP/GFP littermate controls (Figure 9D).In F2 DtTreg mice, other Treg signature proteins were either expressed on a higher proportion of pTreg cells (e.g., Nrp1, CD103, KLRG1) or were expressed at higher levels (GITR) (Figure 9B).Importantly, functional incapacitation of PD-1 in genetargeted mice (91)(92)(93) and human patients treated with blocking Abs (94) can result in overt autoimmune responses, including T1D (94-98), indicating a primarily inhibitory function of PD-1 expression in immune effector cells.However, independent lines of evidence in mice have pointed towards a diabetogenic role of PD-

Discussion
As the functional heterogeneity of pTreg and tTreg cells promises to enable the subset-specific therapeutic manipulation of their activity in various clinical settings, it will be important to define their exact roles in establishing and maintaining peripheral immune homeostasis.The selective ablation of the development of pTreg cells (26,27,102) and tTreg cells, as done here, represents a considerable improvement over previous experiments relying on Foxp3-deficient mice and their reconstitution by adoptive Treg cell transfer, allowing the in vivo consequences to be analyzed under near-physiological conditions, including minimal autoimmune perturbations.In fact, some of the pathology observed in Foxp3deficient mice has been attributed to the enhanced thymic export and peripheral accumulation of Treg cell-like 'wanna-be' CD4 + T cells with self-reactive specificities and distinct pathological properties (9,10), rather than the mere absence of a functional Foxp3 + Treg cell pool.Additionally, some defects of Foxp3-deficient mice (e.g., defective lympho-hematopoiesis) are refractory to adoptive Treg cell therapy, even when total CD4 + T cell populations were used (50).Here we have analyzed how selective tTreg cell paucity, which was achieved by intrathymic tTreg cell ablation while preserving pTreg cell generation, impinges on peripheral immune homeostasis in non-autoimmune and autoimmune-prone mice.Our data in B6.DtTreg mice reveal the ability of pTreg cells to establish immune homeostasis after birth, maintain immune tolerance in young mice, and constrain catastrophic autoimmune responses during aging in the majority of B6.DtTreg mice.Consistently, neonatal transfer of total CD4 + T cell populations, which had been depleted of tTreg cells, ameliorated clinical signs of Foxp3 deficiency in scurfy recipient mice.The manifestation of some mild disease symptoms (moderate growth delay and mild exfoliative dermatitis) can probably be attributed to an initial lag phase after tTreg cell-depleted CD4 + T cell transfer, which is required for seeding and proliferative expansion of pre-formed pTreg cells, and the lymphopenia-driven de novo generation of Foxp3 + pTreg cells (20).
Besides the absence of scurfy-like symptoms, several additional observations in the B6.DtTreg model further support our interpretation that physiologic pTreg cell populations can efficiently constrain autoimmune responses in the absence of tTreg cells.This includes an overall normal size of peripheral lymphoid tissues and T effector cell compartments (numbers, activation state, inflammatory cytokine production, etc.), as well as unperturbed lympho-hematopoiesis, representing a particularly sensitive indicator for the absence of ongoing (auto)immune responses.Many organs of B6.DtTreg mice, which are commonly targeted by severe autoimmune destruction in Foxp3-deficient mice, show no or only mild immune infiltrations not accompanied by any appreciable tissue destruction.Thus, the underlying cause promoting the occurrence of spontaneous deaths from an age of > 7 weeks onwards has remained less clear but may involve the exacerbation of chronic, low-level inflammation in individual organs, such as the thyroid gland or the lungs (Figure 4A), rather than multi-organ autoimmunity observed in Foxp3-deficient models of complete Treg cell deficiency.Interestingly, extending our histopathological analyses of the lungs (Figure 4A) to the upper respiratory tract of DtTreg mice that presented with reduced body weight provided first evidence for unexpected, severe inflammatory changes in the area of the nasal and oral cavities, pointing towards decreased food intake as a possible reason underlying a reduced body weight and morbidity of this particular disease subphenotype (data not shown).
Considering the age-dependent increase in spontaneous deaths (Figure 4B), the abrogation of immune homeostasis in individual DtTreg mice is likely to involve immunological and/or environmental cues (102-104), which are subject to age-related changes.This may include differences in the exposure to antigens derived from the diet and commensal microbiota promoting the physiologic induction of pTreg cells (103, [105][106][107].Our efforts to further analyze the immune events associated with the age-related impairment of peripheral immune homeostasis in DtTreg mice have been hampered by the relatively low incidence of mortality, in conjunction with rapid disease progression.In-depth flow cytometry-based immunophenotyping failed to reproducibly reveal age-related changes in the peripheral immune effector compartments of DtTreg mice, including CD4 + and CD8 + T effector compartments (Figures 5C-E).While this could be taken as an indication for quantitative and/or qualitative changes affecting the pTreg cell compartment, our analyses have not provided any evidence for an age-related reduction of pTreg cell numbers or phenotypic changes in the peripheral pTreg pool.In contrast, we found that the increased pTreg cell population size largely compensated for the numerical impairment of the overall Treg cell pool in adult DtTreg mice (Figures 1D, E; Supplementary Figures S1B, S2), which also holds true for DtTreg mice that were affected by reduced body weight and thymic atrophy (Figure 2B).
At present, we can only speculate on whether the pTreg cell niche in peripheral lymphoid tissues of DtTreg mice is replenished early in life and then maintained by proliferative expansion of pre-formed Treg cells, or whether continuous incorporation of newly formed cells is required to maintain a peripheral pTreg cell pool and immune homeostasis.This raises the possibility that the observed agedependent increase in spontaneous mortality and morbidity is, at least in part, associated with a reduced efficiency in pTreg cell generation.In fact, pTreg cells are thought to be mainly, if not exclusively drawn from initially naïve CD4 + T cells (108).However, rates of thymic export of newly formed CD4 + T cells to peripheral sites of pTreg cell generation continuously decrease during aging and involution of the thymus 110), but also during thymic atrophy due to chronic inflammatory stress (Figure 2B; Supplementary Figure S6C).Consistently, immediate CD25 high Foxp3 − pTreg cell precursors residing in peripheral lymphoid tissues of nonmanipulated mice are strongly enriched among recent thymic emigrants (18).
Overall, our findings are consistent with a scenario, in which pTreg cells in lymphoid tissues of B6.DtTreg mice acquire a highly activated phenotype and increased suppressor function to cope with latent, chronic autoimmune responses due to the absence of tTreg cells.This intricate equilibrium can get out of control even by subtle agerelated immunological and/or microenvironmental changes, which then tip the balance in favor of fatal autoimmunity.This may include changes in the commensal microbiota and qualitative differences among the pTreg cell pool, e.g., reduced rates of pTreg cell de novo generation, in conjunction with proliferative pTreg cell expansion narrowing the TCR repertoire.
This interpretation was further corroborated by the dramatically increased mortality associated with the early onset of severe autoimmune diseases that could be observed in DtTreg mice after only two backcross generations onto the autoimmune-prone NOD background.Here we focused our analysis on pancreatic b cell autoimmunity, as T1D is considered a paradigmatic autoimmune disease for the application of Treg cell-based therapies to prevent or interfere with ongoing autoimmune destruction, although the main regulator(s) of pancreatic b cell autoimmunity hasn't been identified yet.Lastly, Foxp3-deficient NOD mice with a polyclonal CD4 + T cell repertoire fail to present with insulitis and overt diabetes before they succumb at 3 weeks to severe inflammatory infiltration in multiple organs (33), precluding NOD.Foxp3-deficient mice as an experimental model to study the role of Treg cells in the autoimmune b cell protection.Our data show that the acquisition of a small set of Idd risk loci, many of which encode genes with well-known functions in Treg cell biology, is sufficient to precipitate a particularly severe form of autoimmune diabetes in DtTreg mice on a mixed (B6>NOD) background.In this context, it is of interest to note that our complementary studies in DpTreg mice on a (B6>NOD)F5 background haven't provided evidence for a protective role of pTreg cells in the control of b cell autoimmunity (D.M.Z. and K.K., unpublished observation).Our observations in (B6>NOD) hybrid mice with selective tTreg cell paucity, in conjunction with previous experiments in Foxp3.CNS1 -/-with impaired pTreg cell development (34) indicate that tTreg cells are key regulators of b cell autoimmunity in the NOD model.Clearly, future experiments are warranted using Treg cell-subset-deficient mice on a pure NOD background to provide a more definite answer on the role of tTreg and pTreg cells in the control of b cell autoimmunity.
In conclusion, DtTreg and DpTreg mice offer to directly analyze the individual roles of tTreg and pTreg cells, respectively, in the control of immune homeostasis and organ-specific autoimmunity under near-physiologic conditions, which will facilitate future studies on the functional heterogeneity of the mature Treg cell pool.Besides autoimmune diseases, of particular interest will be to dissect their subset-specific contributions to non-immune functions that have recently been attributed to tissue-type Treg cells, which include facilitating homeostasis and regeneration of nonlymphoid tissues.

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FIGURE 1 Selective ablation of tTreg and pTreg cell development in vivo.(A-C) Schematic overview of genetic strategy.(A) Foxp3 RFP/GFP mice.RFP is expressed from an IRES downstream of the endogenous Foxp3 gene in both pTreg and tTreg cells.Restricted activation of BAC.Foxp3 GFP/Cre reporter expression to the thymus results in tTreg cell lineage-specific GFP/Cre activity and induction of gene expression by loxP-flanked STOP cassette excision.(B) DtTreg mice.Ablation of tTreg cells by GFP/Cre-mediated induction of diphtheria toxin A (DTA) expression.(C) DpTreg mice.A Foxp3.STOP cassette precludes pTreg cell development, while tTreg cell development can proceed after GFP/Cre-mediated induction of endogenous Foxp3 gene expression.For this, the Foxp3 IRES-RFP reporter in (A) was replaced by a Cre-activatable Foxp3.STOP cassette.(D-F) Flow cytometry of Treg cells in peripheral lymphoid tissues.(D) Representative dot plots of (left) FSC/SSC-gated and (middle, right) CD4-gated cells from subcutaneous lymph nodes (scLNs) of 21-22-week-old males of indicated mouse lines.Numbers in dot plots represent mean percentages of cells ± SD within the respective gate.(E) Percentages of CD4-gated Foxp3-fluorochrome + Treg cells in scLNs, mesenteric LNs (mLNs), and spleen (SPL) of Foxp3 RFP/GFP mice (pTreg: filled black circles, n = 5; tTreg: filled black squares, n = 5), DtTreg mice (pTreg: open red circles, n = 3), and DpTreg mice (tTreg: open green squares, n = 4).Note that the corresponding cell numbers are shown in Supplementary Figure S1.Symbols and horizontal lines represent individual mice and mean values, respectively.Unpaired t-test: ns, not significant; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, **** p ≤ 0.0001.(F) CD4 + GFP − T cells and CD4 + CD25 + GFP + tTreg cells were FACS-purified from peripheral lymphoid organs of 3-5 males at 10 weeks of age and subjected to flow cytometric analysis of Foxp3, Helios, and Nrp1 expression after intracellular staining using fluorochrome-conjugated mAbs.Numbers in dot plots and histograms represent the percentage of cells within the respective gate.

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FIGURE 9Flow cytometry-based immunophenotyping of pTreg cells in (B6>NOD)F2 DtTreg mice.Expression analysis of selected Treg cell signature proteins on gated Treg cell subsets of I-Ag7 +/+ Foxp3 RFP/GFP mice (total Treg: grey; tTreg: green; pTreg: blue) and pTreg cells of DtTreg mice that were either I-Ag7 +/− (orange) or I-Ag7 +/+ (red).Surface expression of (A) CD25, PD-1, and ICOS, and of (B) Nrp1, GITR, CD103, and KLRG1 on pTreg cells from F2 DtTreg mice, as compared to pTreg cells from F2 Foxp3 RFP/GFP mice.Numbers in histograms indicate mean percentages of cells ± SD within the respective gate or quadrant, with the exception of GITR in (B) showing mean fluorescence intensity (MFI) of the fluorochrome-conjugated mAb (C) Quantification of indicated marker expression based on MFI of fluorochrome-conjugated mAbs.Symbols and bars represent individual mice and mean values ± SD, respectively.(D) Expression of ICOS and PD-1 on pTreg cells from pLNs of Foxp3 RFP/GFP mice and DtTreg mice, as indicated.Numbers in dot plots indicate mean percentages of cells ± SD within the respective quadrant or gate.Note that gated populations of total Treg cells and tTreg cells from Foxp3 RFP/GFP mice were included for comparison.Data are from a single experiment (n = 4).Unpaired t-test: ns, not significant; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.